There is an imperative clinical need for a small diameter vascular graft with good long-term patentcy for arterial reconstruction. The rationale behind our project is that the low patency associated with synthetic small diameter vascular grafts may be caused by the chronic inflammatory response to synthetic biomaterials. The main goal of this proposal is to evaluate the long-term potential of a new type of small diameter (3 mm) tissue engineered vascular graft (TEVG) produced exclusively from cultured vascular cells without any synthetic or exogenous biomaterial. This is the first totally biological TEVG to display a physiological burst strength (greater than 2000 mmHg). TEVGs produced with human cells were grafted for a week in dogs and have shown good preliminary results. The specific aims of this project are to produce a TEVG with cultured porcine cells and to implant it in pigs for long-term evaluation in an autologous setting. Using established tissue culture techniques, endothelial, smooth muscle and fibroblastic cells will be isolated from inbred miniature pigs, characterized and sub-cultured separately in vitro. Some cell lines will be transfected with a reporter gene, selected and sub-cultured. TEVGs will be produced with normal or transfected cells using the same techniques developed with human cells. These will be implanted as carotid and femoral grafts using standard surgical techniques. After implantation, grafts will be regularly monitored by Doppler signaling and angiography. At specified time points (from 2 weeks up to 2 years), grafts will be removed and analyzed for patency, signs of aneurysm or tearing, histological organization, endothelial coverage and inflammation response. Assays will involve immunostaining of specific cell markers and proliferation antigens. In relevant TEVGs, transfected cells will be identified and cell survival rates established for each of the three cell types. This will produce data on survival, migration and proliferation of grafted cells. From these analyses, we will optimize the design of the graft for highest patency rates and mechanical integrity. Furthermore, the mechanical properties of TEVGs will be characterized upon explantation to monitor time-related changes in strength and compliance. This project presents a novel and completely natural approach to tissue engineering in a field where synthetic materials were considered unavoidable. Ultimately, such TEVG would ensure an endless supply of completely biological and autologous small diameter grafts for vascular reconstruction in patients where autologous blood vessels are not available.